HIV enters host cells through several steps, including the formation of an entry complex, the fusion process, and migration toward nucleus for viral replication. The viral envelop gp41-gp120 heterodimers associate into gp41/gp120 trimers to form spikes on the viral surface. Structural studies suggest that a mature HIV particle contains 10-15 spikes each forming a knob with a diameter of 10.5 nm. Binding of CD4 to gp120 leads to conformational changes in a spike that allows gp120 to interact with a chemokine receptor, which completes the formation of an entry complex. Ligation of gp120 to CD4 and a chemokine receptor triggers further structural changes that allow gp41 to insert into the target cell membrane, which triggers the fusion between viral envelop and host cell membrane. Upon the fusion, vesicles that contain virus migrate through cortical actin barrier towards nucleus. In macrophages or T cells where re-organization of actin cytoskeleton is spontaneous and robust, the cortical actin presents little resistance for the HIV migration. Therefore, it appears that chemokine receptor signaling that leads to actin de-polymerization is not essential for HIV infection. In resting T cells, however, it has been demonstrated that CXCR4 signaling mediated de-polymerization of cortical actin matrix is essential for HIV replication. In the past several years, we have investigated CXCR4 and CCR5 signaling in live cells and visualized the gp120-induced interaction between CD4 and CCR5 receptors using fluorescence resonance energy transfer (FRET) imaging techniques. Our studies have provided new insights into the molecular mechanisms underling the earliest step of the formation of HIV entry complex. We found that CD4 and CCR5 localize in different and small microenvironments. CD4 and CCR5 move independently with one another in the membrane before they encounter extracellular ligands. The gp120 variants, JRFL and YU2, that bind extracellular domains of both CD4 and CCR5, induce a clear FRET increase between CCR5-CFP and CD4-YFP, suggesting that our FRET measurements are sensitive enough to monitor the formation of protein complex of gp120, CD4 and CCR5 on the plasma membrane of live cells. The FRET increase induced by gp120 requires cholesterol in the plasma membrane, suggesting that the protein complex of gp120, CD4 and CCR5 forms in a cholesterol-rich microenvironments in live cells. We have also developed a TIRF microscopy method that allows us to visualize single molecules of CD4, CXCR4 or CCR5 receptors that are tagged with YFP. During the development of single molecule analysis, we have assembled an Olympus TIRF (Total Internal Reflection Microscope) system equipped with argon-ion laser excitation and a highly-sensitive CCD camera. This system is able to acquire fluorescence images with required spatial (16 bit) and temporal (the millisecond range) resolution. We have developed a software package for tracking single particles from real-time movies. Our analysis have allowed us to draw conclusions about subpopulations of a receptor, type of diffusion (free or restricted in a microenvironments-the size of microdomains) etc. In a separate study, we visualized the dynamics of cortical actin in live cells using a TIRF microscope. Our previous studies have demonstrated the feasibility of the TIRF imaging for our proposed study.